QuantiFast Multiplex PCR Kits enable fast and reliable quantification of up to 4 cDNA or gDNA targets in a single tube by multiplex, real-time PCR or two-step RT-PCR. Q-bond technology and an optimized master mix promote fast multiplex real-time PCR, not only on fast cyclers with short ramping times, but also on standard cyclers. The combination of a hot start and a unique PCR buffer system in the ready-to-use master mix ensures highly sensitive qPCR on any real-time cycler without the need for optimization. Two kit formats are available: the QuantiFast Multiplex PCR Kit for cyclers that require ROX dye for fluorescence normalization, and the QuantiFast Multiplex PCR +R Kit for all other cyclers. For convenience, the master mix can be stored at 2–8°C.

QuantiFast Multiplex Kits reduce total PCR run time by up to 50% in real-time two-step RT-PCR (40 cycles run; comparison with QuantiTect Multiplex Kits). I: iCycler iQ; L1: LightCycler 480; L2: LightCycler 2.0; A1: ABI PRISM 7900; A2: Applied Biosystems 7500 Fast System; M: Mx3005P. |Duplicate reactions were run on the Applied Biosystems 7500 Fast System using a DNA template mix providing 108 copies of β-actin (data shown in insets) and 106 to 10 copies of RPS27A (a ribosomal protein). The [A] QuantiFast Multiplex PCR +R Kit showed clearly higher sensitivity than the [B] duplex PCR kit from Supplier AII, enabling the cycler in fast-cycling mode to detect 10 copies of target and quantify over 6 log dilutions of template.|Triplex and singleplex PCR were carried out on the LightCycler 480 using the QuantiFast Multiplex PCR +R Kit and Primer Express designed TaqMan assays for [A] RPS27A (a ribosomal protein), [B] GAPDH (a housekeeping gene), and [C] UBC (a housekeeping gene). The template was HeLa cell cDNA (10 ng, 1 ng, or 0.1 ng), and reactions were run in duplicate. The comparable CT values for triplex PCR (colored curves) and singleplex PCR (gray curves) demonstrate the reliability of triplex PCR with the QuantiFast Multiplex PCR +R Kit when analyzing targets of differing abundance.|4-plex, real-time PCR was carried out using the QuantiFast Multiplex PCR +R Kit and Primer Express designed TaqMan assays on the Applied Biosystems 7500 Fast System. Triplicate reactions were run using 10 ng Ramos cell line cDNA.|[A] Q-Bond in QuantiFast Buffer allows the DNA polymerase and primer to bind as a single complex, reducing the annealing time to a few seconds. In addition, the unique buffer composition supports the melting of DNA, reducing denaturation and extension times. [B] Without Q-Bond, the primer and polymerase bind sequentially to the template, increasing annealing time.|QuantiFast Multiplex PCR Kits provide a simple procedure for quantitative, multiplex, real-time PCR. In contrast to current methods, the kits eliminate the need for optimization of the concentrations of primers, Mg2+, and Taq DNA polymerase, even for difficult assays (e.g., assays in which the copy number of the target gene is much smaller than that for the reference gene).|[A] NH4+ ions prevent nonspecific primers from annealing to the template. [B] Synthetic Factor MP, an innovative PCR additive, increases the local concentration of primers at the template. Together with K+ and other cations, Factor MP stabilizes specifically bound primers, allowing efficient primer extension by HotStarTaq Plus DNA Polymerase.|Duplex and singleplex PCR were carried out using the QuantiFast Multiplex PCR +R Kit and assays for the t(8;14) chromosomal translocation and for GAPDH on the Applied Biosystems 7500 Fast System. Quadruplicate reactions were run using genomic DNA from the Ramos cell line as template (twofold dilutions from 10 ng to 0.625 ng). CT values increased linearly by 1 CT value with decrease in template dilution for both the [A] singleplex and [B] duplex reactions, demonstrating the ability of the kit to precisely discriminate between small differences in template amount.|

The special master mix supplied with QuantiFast Multiplex PCR Kits allows rapid setup of multiplex reactions and delivers successful results at the first attempt, providing multiplex PCR data that are comparable with singleplex PCR data (see figure "Comparable results in triplex and singleplex PCR"). The kits can clearly distinguish between small differences in the amount of template. Even with twofold differences in template amount, QuantiFast Multiplex PCR Kits provide accurate quantification of targets of widely differing abundance (see figure "Linear CT values over twofold decreases in template").

QuantiFast Multiplex PCR Kits reduce PCR run times by up to 50%, allowing you to get results significantly faster (see figure "Significantly reduced PCR times"). As little as 10 copies of target can be detected in just 60 minutes (see figure "Sensitive duplex PCR and a wide dynamic range"). You can also greatly increase your sample throughput or efficiently share a cycler with other users. Fast results in multiplex, real-time PCR of up to 4 targets are achieved without compromising performance (see figure "Uncompromised sensitivity").

Amplifying reference and target genes in the same reaction instead of in separate reactions increases the reliability of gene quantification by minimizing handling errors. The QuantiFast Multiplex PCR Buffer includes a balanced combination of K+ and NH4+ ions as well as the unique synthetic Factor MP, which together promote stable and efficient annealing of primers and probes to the nucleic acid template, enabling high PCR efficiency (see figure "Unique PCR buffer"). In addition, HotStarTaq Plus DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products.

Components of 2x QuantiFast Multiplex PCR Kit*

Component

Features

Benefits

HotStarTaq Plus DNA Polymerase

5 min activation at 95ºC

Set up of qPCR reactions at room temperature

QuantiFast Multiplex PCR Buffer

Balanced combination of NH4+ and K+ ions

Specific primer annealing ensures reliable PCR results

Synthetic Factor MP

Reliable multiplexing analysis of up to 4 genes in the same tube

Unique Q-Bond additive

Faster PCR run times, enabling faster results and more reactions per day

Precise quantification on cyclers that require ROX dye. Does not interfere with PCR on any real-time cycler

* Also contains a dNTP mix (dATP, dCTP, dGTP, and dTTP).† ROX dye is either present in the master mix or as a separate solution.

Procedure

QuantiFast Multiplex PCR Kits contain ready-to-use master mixes that eliminate the need for optimization of reaction and cycling conditions. Simply add template DNA and primer-probe sets to the master mix and follow the protocol in the handbook to get fast and reliable results on any real-time cycler. Kits are available with or without ROX passive reference dye in the master mix, enabling use on virtually any real-time cycler (see table). Due to the optimized ROX concentrations, detection of even low copy numbers is achieved through automatic data analysis.

For optimal results in real-time two-step RT-PCR, we recommend synthesizing cDNA using the QuantiTect Reverse Transcription Kit, which provides fast cDNA synthesis in just 20 minutes with integrated removal of genomic DNA contamination.

QuantiFast Probe Assays are predesigned, genomewide assays that use hydrolysis, probe-based detection. They are delivered with the QuantiFast Multiplex PCR Kit for guaranteed results in duplex, two-step real-time RT-PCR.

Applications

QuantiFast Multiplex PCR Kits can be used for multiplex gene expression analysis of cDNA or gDNA targets on any real-time cycler. This includes instruments from Agilent, Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, and Roche. For the Rotor-Gene Q and other Rotor-Gene cyclers, we recommend using the Rotor-Gene Multiplex PCR Kit, which has been specially developed for fast cycling on these instruments.

Feature

Specifications

Applications

Quantification of cDNA or genomic DNA targets in a multiplex real-time PCR